Neuregulin peptides and their use

ABSTRACT

The present invention provides certain neuregulin peptides useful in, for example, methods and compositions comprising preventing, treating or delaying various diseases or disorders.

1. FIELD OF THE INVENTION

This invention relates generally to neuregulin peptides with an enhancedaffinity for the ErbB2 receptor and their use for treating variouscardiac diseases or disorders.

2. BACKGROUND OF THE INVENTION

Cardiac (ventricular) hypertrophy is an important adaptive physiologicalresponse to increased stress or demands for cardiac work. One of theearly cellular changes that occurs after a stimulus for hypertrophy isthe synthesis of mitochondria and expansion of myofibrillar mass (wallthickening) with a proportional increase in the size of individualcells, but no (or minimal) increase in the number of cells.

When the ventricle is stressed, the initial response is an increase insarcomere length. This is followed by an increase in the total musclemass. When the overload is severe; myocardial contractility becomesdepressed. In its mildest form, this depression is manifested by areduction in the velocity of shortening of unloaded myocardium or by areduction in the rate of force development during isometric contraction.As myocardial contractility becomes further depressed, a more extensivereduction in the velocity of shortening of unloaded myocardium occurs,now accompanied by a decline in isometric force development andshortening. At this point, circulatory compensation may still beprovided by cardiac dilation and an increase in muscle mass, which tendto maintain wall stress at normal levels. As contractility fallsfurther, overt congestive heart failure, reflected in a depression ofcardiac output and work and/or an elevation of ventricular end-diastolicvolume and pressure at rest, supervenes.

The transition from hypertrophy to heart failure is characterized byseveral alterations in cellular organization. For example, normalhypertrophic cells have a large size with increased and well organizedcontractile units, as well as strong cell-cell adhesions. In contrast,pathologically hypertrophic cells, which also have large size andaccumulation of proteins, display disorganization of contractileproteins (disarray of sarcomeric structures) and poor cell-celladhesions (disarray of myofibers). Thus, in pathological hypertrophy,the increased size and accumulation of contractile proteins areassociated with disorganized assembly of sarcomeric structures and alack of robust cell-cell interactions.

Heart failure affects approximately five million Americans, and morethan 550,000 new patients are diagnosed with the condition each year.Current drug therapy for heart failure is primarily directed toangiotensin-converting enzyme (ACE) inhibitors, which are vasodilatorsthat cause blood vessels to expand, lowering blood pressure and reducingthe heart's workload. While the percent reduction in mortality has beensignificant, the actual reduction in mortality with ACE inhibitors hasaveraged only 3%-4%, and there are several potential side effects.

ACE inhibitors have also been administered in combination with otherdrugs such as digitalis, which increases the force of the heart'scontractions, and/or a diuretic, which helps relieve the heart'sworkload by causing the kidneys to remove more sodium and water from thebloodstream. However, at least one study demonstrated no difference insurvival associated with the use of digitalis compared with placebo inpatients with Class II-III heart failure. Additionally, diuretics canimprove some symptoms of heart failure but it is not suitable as a soletreatment.

Additional limitations are associated with other options for preventingor treating heart failure. For example, heart transplantation is clearlymore expensive and invasive than drug treatment, and it is furtherlimited by the availability of donor hearts. Use of mechanical devices,such as biventricular pacemakers, are similarly invasive and expensive.Thus, there has been a need for new therapies given the deficiencies incurrent therapies.

One promising new therapy involves administration of neuregulin(hereinafter referred to as “NRG”) to a patient suffering from or atrisk of developing heart failure. NRGs comprise a family of structurallyrelated growth and differentiation factors that include NRG1, NRG2, NRG3and NRG4 and isoforms thereof. For example, over 15 distinct isoforms ofNRG1 have been identified and divided into two large groups, known as α-and β-types, on the basis of differences in the sequence of theiressential epidermal growth factor (EGF)-like domains. NRG-1 isdescribed, for example, in U.S. Pat. Nos. 5,530,109, 5,716,930, and7,037,888; Lemke, Mol. Cell. Neurosci.1996, 7:247-262; Peles and Yarden,1993, Bio Essays 15:815-824, 1993; Peles et al., 1992, Cell 69, 205-216;Wen et al., 1992, Cell 69, 559-572, 1992, Holmes et al., 1992, Science256:1205-1210, Falls et al., 1993, Cell 72:801-815, Marchionni et al.1993, Nature 362:312-8, the contents of which are incorporated byreference in their entireties. NRG-2 is described, for example, in Changet al., 1997, Nature 387:509-512; Carraway et al., 1997, Nature387:512-516; Higashiyama et al., 1997, J. Biochem. 122:675-680, Busfieldet al., 1997, Mol. Cell. Biol. 17:4007-4014 and International Pat. Pub.No. WO 97/09425), the contents of which are incorporated by reference intheir entireties. NRG-3 is described, for example, in Hijazi et al.,1998, Int. J Oncol. 13:1061-1067, the contents of which are incorporatedby reference in their entireties. NRG-4 is described, for example, inHarari et al., 1999 Oncogene. 18:2681-89, the contents of which areincorporated by reference in their entireties.

NRGs bind to the EGF receptor family, which comprises EGFR, ErbB2, ErbB3and ErbB4, each of which plays an important role in multiple cellularfunctions, including cell growth, differentiation and survival. They areprotein tyrosine kinase receptors, consisting of an extracellularligand-binding domain, transmembrane domain and cytoplasmic tyrosinekinase domain. After NRG binds to the extracellular domain of ErbB3 orErbB4, it induces a conformational change that leads to heterodimerformation between ErbB3, ErbB4 and ErbB2 or homodimer formation betweenErbB4 itself, which results in phosphorylation of the receptors'C-terminal domain inside the cell membrane. The phosphorylatedintracellular domain then binds additional signal proteins inside thecell, activating the corresponding downstream AKT or ERK signalingpathway, and inducing a series of cell reactions, such as stimulation ordepression of cell proliferation, cell differentiation, cell apoptosis,cell migration or cell adhesion. Among these receptors, mainly ErbB2 andErbB4 are expressed in the heart.

It has been shown that the EGF-like domains of NRG1, ranging in sizefrom 50 to 64-amino acids, are sufficient to bind to and activate thesereceptors. Previous studies have shown that neuregulin-1β (NRG-1β)canbind directly to ErbB3 and ErbB4 with high affinity. The orphanreceptor, ErbB2, can form heterodimer with ErbB3 or ErbB4 with higheraffinity than ErbB3 or ErbB4 homodimers. Research in neural developmenthas indicated that the formation of the sympathetic nervous systemrequires an intact NRG-1β, ErbB2 and ErbB3 signaling system. Targeteddisruption of the NRG-1β or ErbB2 or ErbB4 led to embryonic lethalitydue to cardiac development defects. Recent studies also highlighted theroles of NRG-1β, ErbB2 and ErbB4 in the cardiovascular development aswell as in the maintenance of adult normal heart function. NRG-1β hasbeen shown to enhance sarcomere organization in adult cardiomyocytes.The short-term administration of a recombinant NRG-1β EGF-like domainsignificantly improves or protects against deterioration in myocardialperformance in three distinct animal models of heart failure. Moreimportantly, NRG-1β significantly prolongs survival of heart failureanimals. These effects make NRG-1β promising as a broad spectrumtherapeutic or lead compound for heart failure due to a variety ofcommon diseases. However, there remains a need for more effective NRGpeptides that can be used in a clinical setting for the prevention,treatment or delaying of heart failure and/or cardiac hypertrophy.

3. SUMMARY OF THE INVENTION

The present invention provides neuregulin peptides having an enhancedaffinity for an ErbB receptor. In some embodiments, the neuregulinpeptides of the invention comprise an EGF domain of neuregulin. In someembodiments, the neuregulin peptides of the invention comprise an EGFdomain of human neuregulin β2 isoform. In some embodiments, theneuregulin peptides having an enhanced affinity for an ErbB receptorcomprise the amino acid sequence of SEQ ID NO:5.

The neuregulin peptides of the invention have an enhanced affinity foran ErbB receptor compared to the affinity of the wild type full lengthneuregulin. In addition to ErbB receptor binding, The neuregulinpeptides can possess one or more other biological activities of a nativeheregulin.

The neuregulin peptides can be prepared according to any techniqueapparent to those of skill. Exemplary techniques for the preparation ofthe neuregulin peptides are described herein. In some embodiments, theneuregulin peptides can be prepared recombinantly. In certainembodiments, the neuregulin peptides can be prepared synthetically, forexample by solution phase or solid phase peptide synthesis.

The present invention also provides nucleic acid-molecules, vectors andhost cells related to the neuregulin peptides of the invention. Anucleic acid molecule of the invention encodes, or is complementary to anucleic acid molecule encoding, a neuregulin peptide of the invention ora fragment thereof. The nucleic acid molecule can be double- orsingle-stranded DNA or RNA. A nucleic acid molecule of the invention canbe inserted into an appropriate vector for propagation and/or expressionof an encoded neuregulin peptide. Such vectors are introduced intosuitable hosts, for example, to allow recombinant production of aneuregulin peptide.

The neuregulin peptides of the invention are useful in a variety oftherapeutic and non-therapeutic applications. In particular, neuregulinpeptides can be used in treating receptor for preventing, treating ordelaying various cardiac diseases or disorders. Accordingly, theinvention encompasses a pharmaceutical composition comprising aneuregulin peptide and related treatment methods.

In another aspect, the invention provides methods for treating heartfailure in a mammal. In certain embodiments, the method comprise thestep of administering the neuregulin peptides of the invention into amammal in need thereof.

In another aspect, the invention provides method s for inducing thephosphorylation of ErbB receptors in a cell. In certain embodiments, themethod comprise the step of contacting the cell with the neuregulinpeptides of the invention.

In another aspect, the invention provides method s for inducing orsustaining activation of the ERK signaling pathway in a cardiac cell. Incertain embodiments, the method comprise the step of contacting thecardiac cell with the neuregulin peptides of the invention.

In another aspect, the invention provides method s for inducing orsustaining activation of the ERK signaling pathway in a cardiac cell. Incertain embodiments, the method comprise the step of contacting thecardiac cell with the neuregulin peptides of the invention.

4. BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the effect of NRG55, NRG57 and NRG59 at differentconcentration on the phosphorylation of AKT in cardiomyocytes. GAPDH wasshowed to compare the amount of protein in each sample.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein, the singular forms “a”, “an”, and “the” mean “at leastone” or “one or more” unless the context clearly dictates otherwise.

As used herein, “epidermal growth factor-like domain” or “EGF-likedomain” refers to a peptide motif encoded by the neuregulin gene thatbinds to and activates ErbB2, ErbB3, ErbB4, or combinations thereof, andbears a structural similarity to the EGF receptor-binding domain asdisclosed in WO 00/64400, Holmes et al., Science, 256:1205-1210 (1992);U.S. Pat. Nos. 5,530,109 and 5,716,930; Hijazi et al., Int. J. Oncol.,13:1061-1067 (1998); Chang et al., Nature, 387:509-512 (1997); Carrawayet al., Nature, 387:512-516 (1997); Higashiyama et al., J. Biochem.,122:675-680 (1997); and WO 97/09425, the contents of which are allincorporated herein by reference. In certain embodiments, EGF-likedomain binds to and activates ErbB2/ErbB4 or ErbB2/ErbB3 heterodimers.In certain embodiments, EGF-like domain comprises the amino acidsequence of the receptor binding domain of NRG-1. In some embodiments,EGF-like domain comprises the amino acid sequence corresponding to aminoacid residues 177-226, 177-237, or 177-240 of NRG-1. In certainembodiments, EGF-like domain comprises the amino acid sequence of thereceptor binding domain of NRG-2. In certain embodiments, EGF-likedomain comprises the amino acid sequence of the receptor binding domainof NRG-3. In certain embodiments, EGF-like domain comprises the aminoacid sequence of the receptor binding domain of NRG-4. In certainembodiments, EGF-like domain comprises the amino acid sequence of AlaGlu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys AspLeu Ser Asn Pro (SEQ ID NO:11), as described in U.S. Pat. No. 5,834,229.

As used herein, an “effective amount” of an active agent for treating aparticular disease is an amount that is sufficient to ameliorate, or insome manner reduce the symptoms associated with the disease. The amountmay cure the disease but, typically, is administered in order toameliorate the symptoms of the disease.

As used herein, “active agent” means any substance intended for thediagnosis, cure, mitigation, treatment, or prevention of disease inhumans and other animals, or to otherwise enhance physical and mentalwell being.

As used herein, “amelioration” of the symptoms of a particular disorderby administration of a particular active agent refers to any lessening,whether permanent or temporary, lasting or transient that can beattributed to or associated with administration of the agent.

As used herein, “treat”, “treatment” and “treating” refer to any mannerin which the symptoms of a condition, disorder or disease areameliorated or otherwise beneficially altered. The effect may beprophylactic in terms of completely or partially preventing a disease orsymptom thereof and/or may be therapeutic in terms of a partial orcomplete cure for a disease and/or adverse effect attributable to thedisease. Treatment also encompasses any pharmaceutical use of thecompositions herein.

As used herein, “vector (or plasmid)” refers to discrete elements thatare used to introduce heterologous DNA into cells for either expressionor replication thereof. Selection and use of such vehicles are wellknown within the skill of the artisan. An expression vector includesvectors capable of expressing DNA that are operatively linked withregulatory sequences, such as promoter regions, that are capable ofeffecting expression of such DNA fragments. Thus, an expression vectorrefers to a recombinant DNA or RNA construct, such as a plasmid, aphage, recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the cloned DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome.

As used herein, “cardiac muscle cell differentiation” means a conditioncharacterized by the decrease in DNA synthesis by more than 10%,inhibition of other factor-stimulated DNA synthesis more than 10%, wellorganized sarcomeric structures and cell-cell adhesions, sustainedactivation of MAP kinases, and enhanced expression of p21^(C1P1).Further discussion is provided in WO00/37095, the contents of which areincorporated herein by reference in their entireties.

As used herein, “ejection fraction” or “EF” means the portion of bloodthat is pumped out of a filled ventricle as the result of a heartbeat.It may be defined by the following formula: (LV diastolic volume—LVsystolic volume)/LV diastolic volume.

As used herein, “fractional shortening” or “FS” means a ratio of thechange in the diameter of the left ventricle between the contracted andrelaxed states. It may be defined by the following formula: (LV enddiastolic diameter —LV end systolic diameter)/LV end diastolic diameter.

As used herein, “heart failure” means an abnormality of cardiac functionwhere the heart does not pump blood at the rate needed for therequirements of metabolizing tissues. Heart failure includes a widerange of disease states such as congestive heart failure, myocardialinfarction, tachyarrhythmia, familial hypertrophic cardiomyopathy,ischemic heart disease, idiopathic dilated cardiomyopathy, myocarditisand the like. The heart failure can be caused by any number of factors,including, without limitation, ischemic, congenital, rheumatic, oridiopathic forms. Chronic cardiac hypertrophy is a significantlydiseased state which is a precursor to congestive heart failure andcardiac arrest.

As used herein, “myocardial infarction” refers to a blockade of acoronary artery or blood flow interruption leading to focal necrosis ofpart of the myocardium caused by severe and persistent ischemia.

As used herein, “organized, or enhanced organization of sarcomeres orsarcomeric structures” means a condition characterized by the straightarray of contractile proteins revealed by immunofluorescent staining ofa-actinin in cardiac muscle cells. The straight array of a-actininproteins in cells can be distinguished by microscopy and its connectedphotography. As used herein, “disorganized or disarray of sarcomeres orsarcomeric structures” means the opposite of the “organized, or enhancedorganization of sarcomeres or sarcomeric structures”

As used herein, “organized, or enhanced organization of cytoskeletonstructures” means a condition characterized by the straight actin fibersrevealed by phalloidin staining of cardiac muscle cells. The straightactin fibers in cells can be distinguished by microscopy and itsconnected photography as exampled in figures of this specification. Asused herein, “disorganized or disarray of cytoskeleton structures” meansthe opposite of “organized, or enhanced organization of cytoskeletonstructures”.

As used herein, “protein” is synonymous with “peptide” or “peptide”unless the context clearly dictates otherwise.

As used herein, “sustained activation of MAP kinases” means that thephosphorylated state of MAP kinases, p42/44, is maintained for at least21 hr in cells. Further discussion is provided in WO00/37095, thecontents of which are incorporated herein by reference.

The terms “synergistic, “synergistic effect” and like are used herein todescribe improved treatment effects obtained by combining one or moretherapeutic agents with one or more retinoic acid compounds. Although asynergistic effect in some fields is meant an effect which is more thanadditive (e.g., 1+1=3), in the field of medical therapy an additive(1+1=2) or less than additive (1+1=1.6) effect may be synergistic. Forexample, if each of two drugs were to inhibit the development ofventricular muscle cell hypertrophy by 50% if given individually, itwould not be expected that the two drugs would be combined to completelystop the development of ventricular muscle cell hypertrophy. In manyinstances, due to unacceptable side effects, the two drugs cannot beadministered together. In other instances, the drugs counteract eachother and slow the development of ventricular muscle cell hypertrophy byless than 50% when administered together. Thus, a synergistic effect issaid to be obtained if the two drugs slow the development of ventricularmuscle cell hypertrophy by more than 50% while not causing anunacceptable increase in adverse side effects.

As used herein “cardiac hypertrophy” means a condition characterized byan increase in the size of individual ventricular muscle cells, theincrease in cell size being sufficient to result in a clinical diagnosisof the patient or sufficient as to allow the cells to be determined aslarger (e.g., 2-fold or more larger than non-hypertrophic cells). It maybe accompanied by accumulation of contractile proteins within theindividual cardiac cells and activation of embryonic gene expression.

In vitro and in vivo methods for determining the presence of ventricularmuscle cell hypertrophy are known. In vitro assays for ventricularmuscle cell hypertrophy include those methods described WO00/37095,e.g., increased cell size and increased expression of atrial natriureticfactor (ANP). Changes in cell size are used in a scoring system todetermine the extent of hypertrophy. These changes can be viewed with aninverted phase microscope, and the degree of hypertrophy scored with anarbitrary scale of 7 to 0, with 7 being fully hypertrophied cells, and 3being non-stimulated cells. The 3 and 7 states may be seen in Simpson etal. (1982) Circulation Res. 51: 787-801, FIGS. 2, A and B, respectively.The correlation between hypertrophy score and cell surface area (μm2)has been determined to be linear (correlation coefficient=0.99). Inphenylephrine-induced hypertrophy, non-exposed (normal) cells have ahypertrophy score of 3 and a surface area/cell of 581 μm2 and fullyhypertrophied cells have a hypertrophy score of 7 and a surfacearea/cell of 1811 μm2, or approximately 200% of normal. Cells with ahypertrophy score of 4 have a surface area/cell of 771 μm2, orapproximately 30% greater size than non-exposed cells; cells with ahypertrophy score of 5 have a surface area/cell of 1109 μm2, orapproximately 90% greater size than non-exposed cells; and cells with ahypertrophy score of 6 have a surface area/cell of 1366 μm2, orapproximately 135% greater size than non-exposed cells. The presence ofventricular muscle cell hypertrophy preferably includes cells exhibitingan increased size of about 15% (hypertrophy score 3.5) or more. Inducersof hypertrophy vary in their ability to induce a maximal hypertrophicresponse as scored by the above-described assay. For example, themaximal increase in cell size induced by endothelin is approximately ahypertrophy score of 5.

As used herein, “suppression of cardiac hypertrophy” means a reductionin one of the parameters indicating hypertrophy relative to thehypertrophic condition, or a prevention of an increase in one of theparameters indicating hypertrophy relative to the normal condition. Forexample, suppression of ventricular muscle cell hypertrophy can bemeasured as a reduction in cell size relative to the hypertrophiccondition. Suppression of ventricular muscle cell hypertrophy means adecrease of cell size of 10% or greater relative to that observed in thehypertrophic condition. More preferably, suppression of hypertrophymeans a decrease in cell size of 30% or greater; most preferably,suppression of hypertrophy means a decrease of cell size of 50% or more.Relative to the hypertrophy score assay when phenylephrine is used asthe inducing agent, these decreases would correlate with hypertrophyscores of about 6.5 or less, 5.0-5.5, and 4.0-5.0, respectively. When adifferent agent is used as the inducing agent, suppression is examinedrelative to the maximum cell size (or hypertrophic score) measured inthe presence of that inducer.

Prevention of ventricular muscle cell hypertrophy is determined bypreventing an increase in cell size relative to normal cells, in thepresence of a concentration of inducer sufficient to fully inducehypertrophy. For example, prevention of hypertrophy means a cell sizeincrease less than 200% greater than non-induced cells in the presenceof maximally stimulating concentration of inducer. More preferably,prevention of hypertrophy means a cell size increase less than 135%greater than noninduced cells; and most preferably, prevention ofhypertrophy means a cell size increase less than 90% greater thannon-induced cells. Relative to the hypertrophy score assay whenphenylephrine is used as the inducing agent, prevention of hypertrophyin the presence of a maximally-stimulating concentration ofphenylephrine means a hypertrophic score of about 6.0-6.5, 5.0-5.5, and4.0-4.5, respectively.

In vivo determination of hypertrophy may include measurement ofcardiovascular parameters such as blood pressure, heart rate, systemicvascular resistance, contractility, force of heartbeat, concentric ordilated hypertrophy, left ventricular systolic pressure, leftventricular mean pressure, left ventricular end-diastolic pressure,cardiac output, stroke index, histological parameters, and ventricularsize and wall thickness. Animal models available for determination ofdevelopment and suppression of ventricular muscle cell hypertrophy invivo include the pressure-overload mouse model, RV murine dysfunctionalmodel, transgenic mouse model, and post-myocardial infarction rat model.Medical methods for assessing the presence, development, and suppressionof ventricular muscle cell hypertrophy in human patients are known, andinclude, for example, measurements of diastolic and systolic parameters,estimates of ventricular mass and pulmonary vein flows.

Hypertrophy may be from any cause which is responsive to retinoic acid,including congenital viral, idiopathic, cardiotrophic, or myotrophiccauses, or as a result of ischemia or ischemic insults such asmyocardial infarction. Typically, the treatment is performed to stop orslow the progression of hypertrophy, especially after heart damage, suchas from ischemia, has occurred. Preferably, for treatment of myocardialinfarctions, the agent(s) is given immediately after the myocardialinfarction, to prevent or lessen hypertrophy.

As used herein, “activity unit” or “1U” means the quantity of standardproduct that can induce 50% maximal reaction. In other words, todetermine the activity unit for a given active agent, the EC50 must bemeasured. For example, if the EC50 for a batch of product was 0.067μg/ml then that would be one unit. Further, if 1 μg of that product isbeing used then 14.93 U (1/0.067) is being used. The EC50 can bedetermined by any method known in the art, including the method employedby the inventors in the Examples below. This determination of theactivity unit is important for quality control of genetically engineeredproducts and clinically used drugs, permits product from differentpharmaceuticals and/or different batch numbers to be quantified withuniform criteria.

In certain embodiments, unit of neuregulin is determined by measuringthe activity of neuregulin through kinase receptor activationenzyme-linked immunosorbant assay (KIRA-ELISA) as described in detail inWO03/099300, and Sadick et al., 1996, Analytical Biochemistry,235:207-14, the contents of which are incorporated by reference in theirentireties. Briefly, the assay measures neuregulin induced ErbB2activation and phosphorylation on the adherent breast carcinoma cellline, MCF-7. Membrane proteins are solubilized via Triton X-100 lysisand the receptor is captured in ELISA wells coated with ErbB2-specificantibodies (e.g., H4) with no cross-reaction to ErbB3 or ErbB4. Thedegree of receptor phosphorylation is then quantified byantiphosphotyrosine ELISA.

5.2 Neuregulin Peptides of the Invention

The present invention provides neuregulin peptides capable of binding anErbB receptor. In some embodiments, neuregulin peptides have an enhancedaffinity for an ErbB receptor. In some embodiments, the neuregulinpeptides of the invention comprise an EGF domain of neuregulin. In someembodiments, the neuregulin peptides of the invention comprise an EGFdomain of human neuregulin β2 isoform. In some embodiments, theneuregulin peptides comprise the amino acid sequence selected from SEQID NOs:3, 5, 7 and 9.

In some embodiments, the neuregulin peptides comprise the amino acidsequence of SEQ ID NO: 3. In preferred embodiments, the neuregulinpeptide consists of the amino acid sequence of SEQ ID NO:3.

In some embodiments, the neuregulin peptides comprise the amino acidsequence of SEQ ID NO: 5. In preferred embodiments, the neuregulinpeptide consists of the amino acid sequence of SEQ ID NO:5.

In some embodiments, the neuregulin peptides comprise the amino acidsequence of SEQ ID NO: 7. In preferred embodiments, the neuregulinpeptide consists of the amino acid sequence of SEQ ID NO:7.

The neuregulin peptides of the invention have an enhanced affinity foran ErbB receptor compared to the affinity of the wild type full lengthneuregulin. In addition to ErbB receptor binding, The neuregulinpeptides can possess one or more other biological activities of a nativeheregulin.

5.2.1 Preparation of the Neuregulin Peptides

The neuregulin peptides can be prepared according to any techniqueapparent to those of skill. Exemplary techniques for the preparation ofneuregulin are described, for example, in U.S. Pat. No. 7,226,907, U.S.Pat. No. 5,367,060, WO 94/026298, WO 03/099300, the contents of whichare hereby incorporated by reference in its entirety.

The neuregulin peptides of the invention can be prepared according toany technique apparent to those of skill. In certain embodiments, theneuregulin peptides of the invention can be prepared synthetically, forexample by solution phase or solid phase peptide synthesis. SeeMerrifield, 1963, J. Am. Chem. Soc. 85:2149; Fields et al., 1990, Int JPept Protein Res. 35:161-214; Fields et al., 1991, Pept Res. 4:95-101;the contents of which are hereby incorporated by reference in theirentirety.

In further embodiments, the neuregulin peptides can be obtained fromnatural sources, recombinant sources or commercial sources. In someembodiments, the neuregulin peptides can be obtained by recombinantlyexpressing and then purifying the neuregulin peptides.

The neuregulin peptides can be purified by any art-known technique suchas high performance liquid chromatography, ion exchange chromatography,gel electrophoresis, affinity chromatography and the like. The actualconditions used to purify a particular neuregulin will be apparent tothose having skill in the art.

5.3 Use of the Neuregulin Peptides

The neuregulin peptides according to the invention can be used accordingto the judgment of those of skill in the art. Exemplary uses aredescribed in for example, in U.S. Pat. No. 7,226,907, U.S. Pat. No.5,367,060, WO 94/026298, WO 03/099300, the contents of each of which areincorporated by reference in its entirety.

The neuregulin peptide are useful in treating a wide range of diseasesor disorders. Exemplary diseases or disorders include cardiovasculardiseases such as heart failure, viral myocarditis or dilated(congestive) cardiomyopathy (DCM), cardiac toxicity, or myocardialinfarction.

In some embodiments, the present invention provides a method fortreating heart failure in a subject in need thereof comprisingadministering to the subject an effective amount of the neuregulinpeptides of the invention.

The neuregulin peptides may be administered to a subject in the form ofa pharmaceutical composition.

The neuregulin peptides can be administered by any route according tothe judgment of those of skill in the art, including but not limited toorally, intravenously, intragastrically, intraduodenally,intraperitoneally or intracerebroventricularly.

In a preferred embodiment, a composition for administration is apharmaceutical composition. Pharmaceutical compositions can comprise aprophylactically or therapeutically effective amount of one or moreprophylactic or therapeutic agents (e.g., a co-complex comprisingneuregulin, or other prophylactic or therapeutic agent), and a typicallyone or more pharmaceutically acceptable carriers or excipients. In aspecific embodiment and in this context, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund'sadjuvant (complete and incomplete)), excipient, or vehicle with whichthe therapeutic is administered. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Water is a preferred carrier whenthe pharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E.W. Martin.

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well-known to those skilled inthe art of pharmacy, and non-limiting examples of suitable excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a patientand the specific active ingredients in the dosage form. The compositionor single unit dosage form, if desired, can also contain minor amountsof wetting or emulsifying agents, or pH buffering agents.

The pharmaceutical compositions may comprise excipients that are wellknown in the art and are listed, for example, in the U.S. Pharmocopia(USP) SP (XXI)/NF (XVI). In general, lactose-free compositions comprisean active ingredient, a binder/filler, and a lubricant inpharmaceutically compatible and pharmaceutically acceptable amounts.Exemplary lactose-free dosage forms comprise an active ingredient,microcrystalline cellulose, pre-gelatinized starch, and magnesiumstearate.

The invention further encompasses administration of pharmaceuticalcompositions and dosage forms that comprise one or more compounds thatreduce the rate by which an active ingredient will decompose. Suchcompounds, which are referred to herein as “stabilizers,” include, butare not limited to, antioxidants such as ascorbic acid, pH buffers, orsalt buffers.

The pharmaceutical compositions and single unit dosage forms can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Such compositions and dosage forms willcontain a prophylactically or therapeutically effective amount of aprophylactic or therapeutic agent preferably in purified form, togetherwith a suitable amount of carrier so as to provide the form for properadministration to the patient. The formulation should suit the mode ofadministration. In a preferred embodiment, the pharmaceuticalcompositions or single unit dosage forms are sterile and in suitableform for administration to a subject, preferably an animal subject, morepreferably a mammalian subject, and most preferably a human subject.

A pharmaceutical composition comprising neuregulin is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, intramuscular, subcutaneous,oral, buccal, sublingual, inhalation, intranasal, transdermal, topical,transmucosal, intra-tumoral, intra-synovial and rectal administration.In a specific embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous, subcutaneous, intramuscular, oral, intranasal or topicaladministration to human beings. In an embodiment, a pharmaceuticalcomposition is formulated in accordance with routine procedures forsubcutaneous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocamne to ease pain at the siteof the injection.

Examples of dosage forms include, but are not limited to: tablets;caplets; capsules, such as soft elastic gelatin capsules; cachets;troches; lozenges; dispersions; suppositories; ointments; cataplasms(poultices); pastes; powders; dressings; creams; plasters; solutions;patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosageforms suitable for oral or mucosal administration to a patient,including suspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a patient.

The composition, shape, and type of dosage forms of the neuregulinpeptides will typically vary depending on their use. For example, adosage form used in the acute treatment of a disorder may contain largeramounts of one or more of neuregulin it comprises than a dosage formused in the chronic treatment of the same disease. Also, thetherapeutically effective dosage form may vary among different types ofcancer. Similarly, a parenteral dosage form may contain smaller amountsof one or more of the active ingredients it comprises than an oraldosage form used to treat the same disease or disorder. These and otherways in which specific dosage forms encompassed by this invention willvary from one another will be readily apparent to those skilled in theart. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., MackPublishing, Easton Pa. (1990).

The neuregulin peptides can be administered by any route according tothe judgment of those of skill in the art, including but not limited toorally, intravenously, intragastrically, intraduodenally,intraperitoneally or intracerebroventricularly.

5.3.1 Dosage and Routes of Administration

The amount of the neuregulin peptides used in the present invention willvary with the nature and severity of the disease or condition, and theroute by which the active ingredient is administered. The frequency anddosage will also vary according to factors specific for each patientdepending on the specific therapy (e.g., therapeutic or prophylacticagents) administered, the severity of the disorder, disease, orcondition, the route of administration, as well as age, body, weight,response, and the past medical history of the patient. Effective dosesmay be extrapolated from dose-response curves derived from in vitro oranimal model test systems.

Exemplary doses of the neuregulin peptides include milligram ormicrogram amounts of neuregulin per kilogram of subject or sample weight(e.g., about 1 microgram per kilogram to about 500 milligrams perkilogram, about 100 micrograms per kilogram to about 5 milligrams perkilogram, or about 1 microgram per kilogram to about 50 micrograms perkilogram). For example, the dosage administered to a patient istypically 0.001 mg/kg to 15 mg/kg of the patient's body weight, based onweight of the active peptide. Preferably, the dosage administered to apatient is between 0.001 mg/kg and 15 mg/kg, 0.005 mg/kg and 10 mg/kg,0.01 mg/kg and 5 mg/kg, 0.001 mg/kg and 4 mg/kg, 0.005 mg/kg and 3mg/kg, 0.01 mg/kg and 2 mg/kg, 0.001 mg/kg and 1 mg/kg, 0.005 mg/kg and0.5 mg/kg, 0.010 mg/kg and 0.2 mg/kg, 0.005 mg/kg and 0.050 mg/kg of thepatient's body weight.

Exemplary doses of the neuregulin peptides also include unit (U) or unitamounts of neuregulin per kilogram of subject or sample weight (e.g.,about 1 U per kilogram to about 5000 U per kilogram, about 10 Umicrograms per kilogram to about 1000 per kilogram, or about 100 U perkilogram to about 500 U per kilogram). For the neuregulin peptides ofthe invention, the dosage administered to a patient is typically 10 U/kgto 1000 U/kg of the patient's body weight, based on weight of the activepeptide. Preferably, the dosage administered to a patient is between 1U/kg and 10,000U/kg, 1 U/kg and 5000 U/kg, 10 U/kg and 5000 U/kg, 10U/kg and 1000 U/kg, 50 U/kg and 2000 U/kg, 50 U/kg and 1000/kg, 50 U/kgand 500 U/kg, 100 U/kg and 1000 U/kg, 100 U/kg and 500 U/kg, 100 U/kgand 200 U/kg, of the patient's body weight.

In general, the recommended daily dose range of the neuregulin peptidesin the methods of the invention for the conditions described herein liewithin the range of from about 0.001 mg to about 1000 mg per day.Specifically, a total daily dose range should be between 0.001 mg perday and 15 mg per day, 0.005 mg per day and 10 mg per day, 0.01 mg perday and 5 mg per day, 0.001 mg per day and 4 mg per day, 0.005 mg perday and 3 mg per day, 0.01 mg per day and 2 mg per day, 0.001 mg per dayand 1 mg per day, 0.005 mg per day and 0.5 mg per day, 0.010 mg per dayand 0.2 mg per day. In managing the patient, the therapy can beinitiated at a lower dose, perhaps about 0.1 μg to about 1 μg, andincreased if necessary up to about 20 μg to about 1000 μg per day aseither a single dose or divided doses, depending on the patient's globalresponse. It may be necessary to use dosages of the active ingredientoutside the ranges disclosed herein in some cases, as will be apparentto those of ordinary skill in the art. Furthermore, it is noted that theclinician or treating physician will know how and when to interrupt,adjust, or terminate therapy in conjunction with individual patientresponse. In certain embodiments, neuregulin is administered in anamount of about 1 U/day to about 10,000 U/day. In some embodiments, itis administered in an amount of about 1 U/day to about 5000 U/day. Insome embodiments, it is administered in an amount of about 10 U/day toabout 2000 U/day. In some embodiments, it is administered in an amountof about 10 U/day to about 1000 U/day. In some embodiments, it isadministered in an amount of about 100 U/day to about 200 U/day.

The neuregulin peptides can also be administered in a dosing schedule or“therapeutic cycle.” Daily dosage of neuregulin in the therapeutic cycleis described in detail above. The therapeutic cycle can last 2 days, 5days, 7 days, 10 days, two weeks, three weeks, four weeks, five weeks,or six weeks.

In certain embodiments, neuregulin is administered daily for each day ofthe therapeutic cycle. In certain embodiments, neuregulin isadministered consecutively for three, four, five, six, seven, eight,nine, ten, eleven or twelve days in a therapeutic cycle. In certainembodiments, in a therapeutic cycle neuregulin is administered on day 1of the cycle and the cycle concludes with one or more days of noneuregulin administration In some embodiments, neuregulin isadministered daily for 3, 5, 7, or 10 days followed by a resting periodin a therapeutic cycle.

6. EXAMPLES 6.1 Example 1 Chemically Synthesis of Neuregulin Peptides

Five neuregulin peptides NRG53 (SEQ ID NO: 3), NRG55 (SEQ ID NO: 4),NRG57 (SEQ ID NO: 5), NRG59 (SEQ ID NO: 6), NRG61 (SEQ ID NO: 1) weresynthesized by GL Biochem (Shanghai) Ltd.

NRG61 comprises the amino acid sequence of Ser His Leu Val Lys Cys AlaGlu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys AspLeu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly AspArg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln(SEQ ID NO:1), which corresponds to amino acids 177-237 of human NRG-1.

The human nucleic acid sequence encoding the fragment is:

agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgcttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc aaatgagtttactggtgatc gctgccaaaa ctacgtaatg gcgagcttct acaaggcgga ggagctgtac cag(SEQ ID NO:2).

Neuregulin peptide EGF53 has the amino acid sequence:

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly GlyGlu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys CysPro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe (SEQID NO:3).

The human nucleic acid sequence encoding the fragment is:

agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgcttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc aaatgagtttactggtgatc gctgccaaaa ctacgtaatg gcgagcttc (SEQ ID NO:4).

Neuregulin peptide NRG55 comprises the amino acid sequence:

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly GlyGlu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys CysPro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe TyrLys (SEQ ID NO:5).

The human nucleic acid sequence encoding the fragment is:

agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgcttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc aaatgagtttactggtgatc gctgccaaaa ctacgtaatg gcgagcttct acaag (SEQ ID NO:6).

Neuregulin peptide NRG57 comprises the amino acid sequence:

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly GlyGlu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys CysPro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe TyrLys Ala Glu (SEQ ID NO:7).

The human nucleic acid sequence encoding the fragment is:

agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgcttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc aaatgagtttactggtgatc gctgccaaaa ctacgtaatg gcgagcttct acaaggcgga g (SEQ ID NO:8).

Neuregulin peptide NRG59 comprises the amino acid sequence:

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly GlyGlu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys CysPro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe TyrLys Ala Glu Glu Leu (SEQ ID NO:9).

The human nucleic acid sequence encoding the fragment is:

agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg aggggagtgcttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgccc aaatgagtttactggtgatc gctgccaaaa ctacgtaatg gcgagcttct acaaggcgga ggagctg (SEQ IDNO:10).

Each peptide was separately dissolved in buffer 1 (0.1M Na₂HPO₄, 0.1 Mcitric acid, 6M Urea, 1 mM EDTA-Na₂, 5 mM DTT, pH 8.0) at 1 mg/ml, thepeptide solution was then left at room temperature for 1 hour. lmlsample solution was mixed with 9 ml buffer 2 (0.01M Na₂HPO₄, 0.01Mcitric acid, 1 mM EDTA-Na₂, 0.5 mM GSSG, 0.5 mM GSH, pH 8.0) so thefinal protein concentration was 0.1 mg/ml. The solution was then stirredat room temperature for 90 minutes before left at 4° C. for 16 hours.The sample was then loaded onto a G25 column and collected in buffer 3(20 mM Na₂HPO₄, 20 mM NaH₂PO₄, pH 6.0).

6.2 Example 2 Receptor Bindings of Chemically Synthesized FiveNeuregulin Peptides

MCF-7 cells was harvested, counted, pelleted and resuspended into DMEM(with 10% serum and 9 μg/ml insulin) at 5×10⁴ cells/ml. 100 μl cellsuspension was added to each well of 96 well plate and the plate wasincubated at 37° C. overnight. The cells were then washed 3 times withPBS and grew in serum free DMEM for another 24 hours.

ErbB2 antibody H4 (Zensun, anti-ErbB2 monoclonal antibody) was dilutedto 6 μg/ml by coating buffer (50 mM Na₂CO₃-NaHCO₃, pH9.6), and added to96 well plate 50 μl/ well. The plate was left at 4° C. overnight to coatwith antibody.

DMEM was sucked away from the starved MCF-7 cells, and 100 μl serialdilutions of NRG, NRG53, NRG55, NRG57, NRG59 or NRG61 in DMEM were addedto each well separately. DMEM was added to two wells as blank. The platewas incubated at 37° C. for 20 min. The cells were washed once with PBSbefore adding 100 μl/ well lysis buffer (50 mM Hepes, pH 8.0, 150 mMNaCl, 2 mM sodium orthovanadate, 0.01% thimerosal, 1% Triton X-100 andone protease inhibitor cocktail tablet per 25 ml solution) and lysing at4° C. for 30 min. The plate was then shaken gently to completely lyseand remove cells from the plate and centrifuged at 15000 rpm for 15 min.

The plate with coating antibody was washed five times with washingbuffer (10 mM PBS, pH7.4, 0.05% Tween 20) before adding 200 μl/well of5% nonfat milk in washing buffer. The plate was incubated at 37° C. for2 hours before washed again 3 times with washing buffer.

A 90 μl solution of lysed cells was drawn from each well in cultureplate and transferred to corresponding well in coated plate. Followingincubation at 37° C. for 1 hour, the coated plate with cell lysis waswashed again 5 times with washing buffer and treated with 100 μlsuitable concentration of horseradish peroxidase (HRP) conjugatedanti-phosphotyrosine monoclonal antibody (Santa Cruz Biotechnology) at37° C. for 1 hour. After the plate was washed again 5 times with washingbuffer, 100 μl freshly prepared HRP substrate solution [50 mM citricacid, 100 mM Na₂PO₄, pH 5.0, 0.2 mg/ml 3,3′,5,5′-tetramethylbenzidine(TMB), 0.003% H₂O₂] was added to each well before the plate wasincubated at 37° C. for 10 min. Finally 50 μl of 2N H₂SO₄ was added toeach well to destroy HRP activity. The OD value at 450 nm for each wellwas read on a microplate reader (BIO-RAD Model 550), and EC50 was theconcentration of neuregulin peptides which achieved 50% of maximum ODvalue. The lower the EC₅₀, the higher the receptor binding affinity ofthe neuregulin peptides.

The EC₅₀ of NRG (full length wild type neuregulin β2 peptide) NRG53,NRG55, NRG57, NRG59 and NRG61 was shown in Table 1.

TABLE 1 EC₅₀ of NRG, NRG53, NRG55, NRG57, NRG59 and NRG61 sample EC50(μg/ml) NRG 0.2772 NRG53 0.814 NRG55 0.0492 NRG57 0.9783 NRG59 0.4605NRG61 1.439

As shown in table 1, EC₅₀ of NRG55 is much lower than that of NRG.

6.3 Example 3 Effect of Chemically Synthesized Neuregulin Peptides onthe Phosphorylation of AKT in Cardiomyocytes

To study the effect of synthesized peptides on ErbB2 and ErbB4 signalingin cardiomyocytes, cardiac cells extracted from left ventricles ofneonatal rats were used.

Cardiac cells were grown in media with serum for 2 days. After the cellsreached about 80% confluence, the media was changed to serum free media.After another 24 hours, different amounts of synthesized neuregulinpeptides or neuregulin were added into separate wells containing thecells for 20 minutes. The media was then aspirated and gel loadingbuffer was added to lyse the cells. The sample was then harvested andloaded into a separate well of gel for electrophoresis and western blotanalysis.

FIG. 1 shows the effect of the neuregulin peptides at differentconcentration on AKT phosphorylation. As shown in FIG. 1, NRG57 andNRG59 almost have the same effect as NRG, NRG55 has much stronger effectthan NRG. The result suggests that NRG55 can be a more effective peptidefor treating cardiovascular disease.

The scope of the invention is not limited by the description of theexamples. Modifications and alterations of the present invention will beapparent to those skilled in the art without departing from the scopeand spirit of the present invention. Therefore, it will be appreciatedthat the scope of this invention is to be defined by the appendedclaims, rather than by the specific examples which have been presentedby way of example.

1. A neuregulin peptide consisting essentially of the amino acidsequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9.
 2. Theneuregulin peptide of claim 1 , wherein the neuregulin peptide consistsof the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 orSEQ ID NO:9.
 3. A pharmaceutical composition comprising a neuregulinpeptide consisting essentially of the amino acid sequence of SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9 and a pharmaceuticallyacceptable carrier, excipient, or diluent.
 4. The pharmaceuticalcomposition of claim 3, wherein the neuregulin peptide consists of theamino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ IDNO:9.
 5. A method for treating heart failure in a subject in needthereof comprising administering to the subject an effective amount of aneuregulin peptide consisting essentially of the amino acid sequence ofSEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9.
 6. The method ofclaim 5, wherein the neuregulin peptide consists of the amino acidsequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9.
 7. Themethod of claim 5, wherein the subject is human.
 8. The method of claim5, wherein the neuregulin peptide is administered intravenously.